Investigation of Zona Pellucida hardening with Atomic Force Microscopy and nonlinear optimization

The Zona Pellucida (ZP) is the extracellular coat surrounding mammalian oocytes. The so called "zona hardening" has a key role in the fertilization process as it produces a block of polyspermy also through an increase of the stiffness of the membrane. The full comprehension of the mechanisms involved in the structural reorganization of the ZP leading to mechanical hardening as well as a correct estimation of its elastic properties is still lacking. In this study, mechanical properties of the ZP membranes extracted from mature and fertilized bovine oocytes were investigated with Atomic Force Microscopy nanoindentation measurements. Both inner and outer sides of the fertilized oocyte's ZP were characterized in order to investigate the propagation of the zona hardening through the thickness of the membrane. This work proposes the application of a hybrid procedure combining experimental measurements, Finite Element analysis and optimization algorithms to analyze the indentation curves.